The invention generally relates to compositions of biologically active components useful for therapeutic applications, such as cancer therapy, and more particularly to compositions including small peptides having (or mimicking) TGFxcex2, activity and collagen receptor agonists.
Cancer is a cellular proliferative disease that is characterized by failure at the level of DNA of normal regulation of growth and/or differentiation. Generally, after an initiating event, there are two stages of the disease. The first is tumorigenesis, the establishment of a cancerous growth. This amplification of cancerous cell populations is supported by increased angiogenesis, which nurtures the growth by enhancing vascular perfusion. Tumorigenesis and angiogenesis seem always to occur together. Later in the natural history of the disease metastasis, in which the cancer spreads to other tissue sites, often occurs. Metastasis results from release and migration of aberrant cells from the primary site of tumorigenesis and their subsequent attachment at distal sites where the processes of tumorigenesis and angiogenesis begin anew.
The major pathobiological processes in tumorigenesis and metastasis can be classified into three categories: cell proliferation leading to cancerous cell amplification and angiogenic cell propagation; integrin-mediated processes of cell attachment and migration, crucial components of both angiogenesis and metastasis; and metalloproteinase-mediated processes that underlie both the release of cancer cells and angiogenesis. Metalloproteinases release aberrant cells from their connective tissue anchorage, facilitating metastatic migration. Angiogenesis depends upon metalloproteinases to clear a path for migrating cells at the advancing capillary front.
Most current approaches to cancer therapy, such as standard chemotherapy and irradiation, are attempts to kill cancer cells. However, the discovery of peptides that inhibit proliferation of cancer cells or inhibit endothelial cell proliferation have raised hopes for new therapeutic modalities. For example, members of the transforming growth factor p family (TGF-xcex2) are among the peptides known to have a number of biological activities related to tumorigenesis (including angiogenesis) and metastasis. TGF-xcex2 inhibits the proliferation of many cell types including capillary endothelial cells and smooth muscle cells. TGF-xcex2 downregulates integrin expression (xcex11xcex21, xcex12xcex21, xcex1xcex1vxcex23 involved in endothelial cell migration). Integrins are involved in the migration of all cells, including metastatic ones. TGF-xcex2 downregulates matrix metalloproteinase expression needed for both angiogenesis and metastasis. TGF-xcex2 induces plasminogen activator inhibitor, which inhibits a proteinase cascade needed for angiogenesis and metastasis. TGF-xcex2 induces normal cells to inhibit transformed cells.
Transforming growth factor-xcex2s were originally named for their ability to transform normal fibroblasts to cells capable of anchorage-independent growth. The effects of TGF-xcex2s on cells are generally classified as proliferative and non-proliferative. As originally established with the first experiments on fibroblasts, TGF-xcex2s are bona fide growth factors. Two important cell types in which proliferation is enhanced by TGF-xcex2 are osteoblasts and Schwann cells of the peripheral nervous system. However, in many cells, TGF-xcex2s are potent inhibitors of cell proliferation. This negative growth control may be the regulatory mechanism that checks regeneration of certain tissues and may play a role in the initiation of carcinogenesis.
The most important non-proliferative function of TGF-xcex2s are in enhancing the formation of extracellular matrices. Although this is achieved primarily through the increased transcription of both collagen and fibronectin, the inhibition of the proteases from degrading the matrix also contributes to its stability. Degradation of the extracellular matrix is inhibited by the decrease in the secretion of the proteases themselves and the simultaneous increase in the levels of protease inhibitors.
Because of the wide applicability of TGF-xcex2s in clinical therapies, they have been the focus of much research. Although much of the research involved in vitro uses, recent in vivo studies have confirmed some of the more promising in vitro effects.
The natural members of the transforming growth factor-xcex2family range upwards of 25 KDa molecular weight. Clinical uses of growth factors, including TGF-xcex2s, may be limited because of their size, which can cause immune responses. For example, human TGF-xcex21 is a 25,000 dalton homodimeric protein. In addition to possible adverse immunological responses, large proteins are not often the best candidates for drugs because of the difficulties in administration and delivery.
Consequently, small peptide mimics of a natural growth factor such as TGF-xcex2 would be desirable. It would also be advantageous to have small peptides mimicking the biological activity of a large, natural protein such as TGF-xcex2 since small peptides on a mole per mole basis would require much smaller net amounts for administration, and topical applications would be more feasible. Also, quite small peptides would tend to have little or no adverse immunological responses, and could be synthesized easily using simple peptide chemistry procedures.
As earlier noted, tumoregenesis and metastasis are integrin-mediated processes of cell attachment and migration. Further, matrix metalloproteinase 1 (MMP-1) processes underlie both the release of cancer cells and angiogenesis. Thus, inhibitors of MMP-1 should interfere with angiogenesis, since it is dependent on the lysis of collagenous matrices in the release of migrating cells in metastasis.
Several recent reports have suggested amelioration of cancer by synthetic versions of endogenous peptides: Cao et al., xe2x80x9cExpression of Angiostatin cDNA in a Murine Fibrosarcoma Suppresses Primary Tumor Growth and Produces Long-term Dormancy of Metastases,xe2x80x9d J. Clin. Invest., 101, (1998), 1055-1063; Boehm et al., xe2x80x9cAntiangiogenic Therapy of Experimental Cancer Does Not Induce Acquired Drug Resistance,xe2x80x9d Nature, 390 (1997), 404-407; and Wu et al., xe2x80x9cSuppression of Tumor Growth with Recombinant Murine Angiostatin,xe2x80x9d Biochem. Biophys. Res. Commun., 236(1997), 651-654.
In one aspect of the present invention, compositions suitable for pharmaceutical administration are provided in which one compound is a small peptide mimic of TGF-xcex2. More preferably, pharmaceutical compositions of the present invention are formulated as combinations of two components, wherein one component includes a peptide mimic for TGF-xcex2 and the other component includes compounds that are structurally or biologically analogous to a small region of collagen and mimic the conformation recognized by collagen binding species.
A feature for TGF-xcex2 activity believed critical is the peptide""s ability to adopt a particular structure, when bound to a TGF-xcex2 receptor, that places certain side-chain functional groups in the appropriate relative positions and orientations. The TGF-xcex2 mimics of the present invention are sometimes herein referred to as xe2x80x9cxcex2-bend peptides,xe2x80x9d or xe2x80x9cextended-bend peptides.xe2x80x9d The appropriate functional groups are represented, in many cases, within the following initial amino acid sequence, AAi-AAi+1-AAi+2 wherein AA, is alanine, asparagine, or leucine, AAi+1 is valine or isoleucine, and AAi+2 is alanine. Of particular importance is the relatively positioning of the side-chains of AAi+1 and AAi+2. The correct positioning of these amino acids can be achieved if AAi+1 and AAi+2 are in either of two backbone conformations: a xcex2-bend or an xe2x80x9cextended-bendxe2x80x9d conformation with the backbone (xcfx86,xcexa8) angles of AAi+1 equal to approximately (xe2x88x9260, +135) and those of AAi+2 equal to approximately (xe2x88x9260, xe2x88x9245). This sequence is often either immediately followed by or has proximal thereto an amino acid with a hydrogen bond acceptor-containing side-chain. Because the amino acid residue with a hydrogen bond acceptor-containing side-chain does not necessarily have to be immediately followed by, that is adjacent to AAi+2, it is referred to as AAi+n where n is an integer equal to or greater than three. If n is greater than 3, then nxe2x88x923 (xe2x80x9cn minus 3 xe2x80x9d) amino acid residues would be between AAi+2 and AAi+n in the peptide sequence.
For example, the TGF-xcex2 mimics may include the sequence AAi-AAi+1-AAi+2-AAi+3 where AAi through AAi+2 are as before described and AAi+3 is an amino acid residue with a hydrogen bond acceptor-containing side-chain (and is glutamic acid, aspartic acid, glutamine, or asparagine). Another example is the sequence AAi-AAi+1-AAi+2-AAi+3-AAi+4 where the residue with the hydrogen bond acceptor-containing side-chain is not immediately adjacent, but instead is proximal to, the initial sequence. In this case, AAithrough AAi+2 are as before, AAi+3 is any suitable amino acid, and AAi+4 may be glutamic acid, aspartic acid, glutamine, or asparagine. Yet another example is the sequence AAi-Ai+1-AAi+2-AAi+3-AAi+4-AAi+5. Here, AAi through AAi+2 are as before, AAi+3 and AAi+4 are suitable amino acids, and AAi+5 may be glutamic acid, aspartic acid, glutamine, or asparagine.
The original peptide discovered to have TGF-xcex2 activity has been named xe2x80x9ccytomodulinxe2x80x9d and has the sequence A-N-V-A-E-N-A (SEQ ID NO: 1). Cytomodulin when added to cells in culture in the concentration range 10xe2x88x929 to 10xe2x88x926 M (1.4 pg/mil to 1400 pg/mil), elicits certain highly specific TGF-xcex2 effects in several different cell types. For example, among the effects observed is the inhibition of DNA synthesis in Mv-1-Lu mink lung epithelial cells, the growth and colony formation by NRK-49 F fibroblasts in soft agar, and the induction of increased expression of type I collagen in primary cultures of neo-natal human dermal fibroblasts. Moreover, results with human osteogenic sarcoma (HOS) cell line indicate that cytomodulin also may be a mimic for other members of the TGF-xcex2 superfamily, such as bone morphogenic proteins (BMPs) and osteogenic protein (OPs), as evidenced by its ability to specifically stimulate markers (alkaline phosphatase and osteonectin) characteristic of the osteoblast phenotype.
Compositions of the subject invention preferably further include a collagen receptor agonist. Particularly preferred such an agonist binds with high affinity with cell surface receptors for collagen and inhibits the attachment and migration of a variety of cells on collagen. These compounds contain a sequence cleaved by MMP-1, and peptides based upon the sequence contained therein are good substrates of MMP-1. Conformationally-restricted compounds capable of serving as allosteric locks or irreversibly binding ligands for receptors and for MMP-1 have potential for cancer therapy, as they are expected to block the migration of smooth muscle cells and attachment of monocytes and other cells by occupying integrin receptors, and to interfere with angiogenesis within developing tumors by inhibiting the attachment and migration of endothelial cells.